Insertion of PsaK into the thylakoid membrane in a "horseshoe" conformation occurs in the absence of signal recognition particle, nucleoside triphosphates, or functional Albino3

The photosystem I subunit PsaK spans the thylakoid membrane twice, with the N and C termini both located in the lumen. The insertion mechanism of a th ylakoid membrane protein adopting this type of topology has not been studie d before, and we have used in vitro assays to determine the requirements fo r PsaK insertion into thylakoids. PsaK inserts with high efficiency and we show that one transmembrane span (the C-terminal region) can insert indepen dently of the other, indicating that a "hairpin"-type mechanism is not esse ntial. Insertion of PsaK does not require stromal extract, indicating that signal recognition particle (SRP) is not involved. Removal of nucleoside tr iphosphates inhibits insertion only slightly, both in the presence and abse nce of stroma, suggesting a mild stimulatory effect of a factor in the tran slation system and again ruling out an involvement of SRP or its partner pr otein, FtsY. We, furthermore, rind no evidence for the involvement of known membrane-bound translocation apparatus; proteolysis of thylakoids destroys the See and Tat translocons but does not block PsaK insertion, and antibod ies against the Oxa1/YidC homolog, Alb3, block the SRP-dependent insertion of Lhcb1 but again have no effect on PsaK insertion. Because YidC is requir ed for the efficient insertion of every membrane protein tested in Escheric hia coli (whether SRP-dependent or -independent), PsaK is the first protein identified as being independent of YidC/Alb3-type factors in either thylak oids or bacteria. The data raise the possibility of a wholly spontaneous in sertion pathway.